HCoV VACCINE FOR IMPROVING IMMUNITY AGAINST SARS-COV-2 INFECTION

ABSTRACT

Embodiments include a method of using inactivated human cold coronaviruses (HCoVs) particularly HCoV-299E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1, alone or as a booster, for the immunization against SARS-CoV-2 infections. Vaccine embodiments further comprise HCoV virus envelope subunits which may be in the form of virus-like spheroids (VLS).

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims priority as a divisional to U.S. patentapplication Ser. No. 16/989,796 filed Aug. 10, 2020.

TECHNICAL FIELD

The present invention in an embodiment relates to a method of usinginactivated human cold coronaviruses (HCoVs) vaccine, alone or as abooster, for the immunization against SARS-CoV-2 infections. PreferredHCoVs are selected from at least one of HCoV-299E, HCoV-OC43, HCoV-NL63and HCoV-HKU1, and preferably selected from a plurality of such group,and most preferably 3 or all four. In a preferred embodiment the vaccinecomprises HCoV virus envelope subunits. In a particularly preferredembodiment the vaccine comprises HCoV virus envelope protein in avirus-like sphere (VLS). One method for inactivation of HCoVs, comprisesthe steps of exposure to copper atoms followed by hydrogen peroxidetreatment. Also provided is a vaccine for immunization againstSARS-CoV-2 infection comprising at least one unique protein epitope, orgenetic instructions to make such one unique protein epitope, from aplurality of the group of HCoV-299E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1,such unique protein epitope being unique to all other HCoVs in suchgroup, wherein the unique epitopes have epitope homology with SARS-CoV-2of greater than or equal to 60%. Such HCoV vaccines may be used as abooster, for example, post-immunization with a vaccine designed toproduce a specific SARS-CoV-2 protein, such as spike protein, to providefor longer lasting effective T-cell memory.

BACKGROUND

A new coronavirus, designated SARS-CoV-2, has ravaged the world sinceDecember 2019. This virus first jumped into humans in Wahun, HubeiProvince, China, and then quickly spread across the world. SARS-CoV-2had never before the infections in China been reported in humans. OnJan. 31, 2020, the Secretary of HHS issued a declaration of publichealth emergency related to COVID-19. On Mar. 11, 2020 the WHO declaredthe outbreak of SARS-CoV-2 to be a pandemic. It is estimated thatsomewhere around 20 percent of infected individuals may develop seriousconsequences. It is believed that the main cause of transmission is byrespiratory droplets, albeit other routes have been hypothesized such asdirect human to human contact and fecal to oral contamination. Theincubation period of the disease is believed to be 14 days.

Coronaviruses (CoVs) are among the largest known group of viruses in thefamily Coronaviridae and order Nidovirales. SARS-CoV-2 belong to theBetacoronavirus genus. It has a genome size of approximately 30kilobases, and is Baltimore class IV positive-sense single-stranded RNAvirus. Four structural proteins are encoded in the RNA, spike (S)protein, envelope (E) protein, membrane (M) protein, and nucleocapsid(N) protein. Betacoronaviruses are spherical or pleomorphic in shapehaving an average diameter of about 125 nm. Cornoaviruses are enveloped,that is, have lipid layers.

Recent studies suggest that vaccines can be manufactured to provideprotective humoral and cell-mediated immune response against SARS-CoV-2.Many different types of vaccines have been proposed for the immunizationagainst SARS-CoV-2. Vaccine clinical development follows the generalpathway as for drugs and other biologics. A sponsor who wishes to beginclinical trials with a vaccine must submit an Investigational New DrugApplication (IND) to the FDA. The IND describes the vaccine, its methodof manufacture and quality control tests for release. Such also includesinformation about the vaccine's safety and ability to elicit aprotective immune response (immunogenicity) in animal testing, as wellas proposed clinical protocol for studies in humans.

Although only a few recombinant technology vaccines have been brought tocommercial market to date, of the 164 candidate vaccines reported by theWorld Health Organization on Jul. 28, 2020 (World Health Organization,Draft Landscape of COVID019 Candidate Vaccines, 31 Jul. 2020), only 11(7 percent) are employing inactive virus or live attenuated virus, thestandard methods of vaccine production (8 using whole or submitinactivated virus, 3 using live attenuated virus) for decades. Of the 25candidate vaccines in clinical evaluation as of Jul. 28, 2020, five (20percent) are inactive viruses, suggesting that such technology can stillbe time competitive in emergencies to vaccines employing recombinanttechniques.

SARS-CoV-2 vaccines that employ bioengineering comprise 93 percent ofvaccine candidates reported by the World Health Organization on Jul. 28,2020. While not mutually exclusive, the present technologies arecharacterized by the WHO in certain set categories.

Replicating or non-replicating viral vector vaccines typically splice ingenetic instructions for the S protein of SARS-CoV-2 into a relativelyharmless virus (such as Adenovirus Type 5 Vector, or ChAdOx1-S) to causethe cells to mass-produce the protein, with the immune system developingantibodies thereto. Twenty-four of the 164 vaccine candidates as of Jul.28, 2020 are non-replicating vaccines, while 18 are replicating virusvaccines. To date no non-replicating viral vectors have been licensedfor immunity commercially. Three of the 25 candidate vaccines inclinical evaluation as of Jul. 28, 2020 were of the non-replicatingviral vector type.

Virus like particle vaccines introduce particles that have enoughforeign immunogen to cause the body to produce an immunologicalresponse. Virus-like particles are multiprotein structures that mimicthe organization and conformation of authentic native viruses but lackthe viral genome, and therefore are not infectious. Structurally simpleVLPs composed of no more than one or two proteins are normally expressedin bacteria or yeast (with for yeast-based VLPSs vaccines approved for anumber of vaccine products). Eukaryotic expression systems, insect andmammalian cells, have been extensively used for both intracellular andsecretory production of enveloped VLPs because of their more complexpost-translational modification system and that they support mostaspects of the virus life cycle. Plant expression systems may also beused. Four commercially available prophylactic VLP vaccines arecurrently available, GlaxoSmithKiline's Engerix (hepatitis B Virus andCervarix (human papillomavirus), Merck and Co., Inc.s Recombivax HB(heptatits B Virus) and Gardasil (human papillomavirus). Twelve out ofthe 164 vaccine candidates identified by the WHO (Jul. 28, 2020) makeuse of such technology with one of the 25 vaccines in clinical trial asof Jul. 28, 2020 being of this type.

Another technique characterized by the WHO is the protein subunitvaccine, wherein parts of the S protein or Receptor-Binding Domain (RBD)thereof are used to induce an immune response that primes the body toattack the virus. Fifty-five out of the 164 vaccine candidates as ofJul. 28, 2020 are of this type, with 7 out of the 25 in clinical trialsas of Jul. 28, 2020 being of this type.

WHO also characterizes numerous vaccines as RNA or DNA based. Thesevaccines make use of the sequenced SARS-CoV-2 genome (RNA based).Plasmid DNA or RNA, for example, mRNA or viral replicon, are used in thenucleic acid based approaches. DNA plasmid vaccines work by transferringthe genetic blueprint to RNA in the cell machinery, which makes spikeantigens. When the genetic material is injected into the body it istaken up by cells that being to produce the S protein (most) or otherprotein, triggering an immune response. RNA is often encased in a lipidcoat so it can enter the cell. mRNA vaccines use the host body toproduce the viral proteins, thereby bypassing the hassle of producingpure viral proteins, which is thought to sometimes save months or yearsin standardization and ramping up for mass production. mRNA has theadvantage that it cannot become part of a person's chromosomes. Theadvantage of such vaccines is reducing the task of isolating pure viralproteins. As of Jul. 28, 2020, WHO characterizes 21 vaccines asRNA-based vaccines, and 15 vaccines as DNA based vaccines, with 4 RNAvaccines and 4 DNA vaccines in clinical trials.

One other vaccine that is not specified in the WHO Jul. 28, 2020 list,and does not nicely fit into the classification scheme set forththerein, is a vaccine being developed by Immunor AS, Biovacc-19 (See,Sorensen et al., Biovacc- 19: A Candidate Vaccine of Covid-19(SARS-CoV-2) Developed from Analysis of its General Method of Action forInfectivity, Pre-publication print QRB Discovery, DOI 10.017). Thisvaccine is based on the understanding that antibodies can only recognize5-6 amino acids. Biovacc-19 is said to be a peptide vaccine designed todevelop antibodies to those parts of the SARS-CoV-2 spike protein whichare engaged in binding and infecting cells and which are non-human-likein nature. Peptide strings of total length of 30 to 36 amino acids areplaced into scaffolds and used to produce an immunological effect.

Inactivated virus vaccines follow the protocol pioneered by Jonas Salkin the early 1950s in the development of the Salk injectable poliovaccine (and later hepatitis A and rabies vaccine), that is they takelive viruses and kill them so they cannot replicate. They aredifferentiated from attenuated vaccines that are created by reducing thevirulence of a pathogen, but still keeping it viable (attenuated virusesare typically produced by passage by passage of the virus through aforeign host multiple times until the virus remains active but fairlyharmless). Inactive virus vaccines may be known as whole Killed Virusvaccines (WKV) when the entire virus is used in making the vaccine.Albeit, most researchers seeking to rapidly produce a SARS-CoV-2 vaccinerejected what until now has been the gold standard of availablevaccines, inactivated and attenuated vaccines, still 1 in 5 havingindicated by WHO to have entered clinical trials as of Jul. 28, 2020 areproduced by simple inactivation processes. Indeed, out of the sixcandidate vaccines in Phase 3 as of Jul. 28, 2020, three (fifty percent)are inactivated virus vaccines.

Three major factors have led groups to recombinant technology, first abelieved speed in which a consistent vaccine may be made manufactured ascompared to standard techniques, second the commercially cheaperexpenditure to produce recombinant vaccines, and lastly a belief thatinactivated and attenuated viruses include unnecessary antigenic loadthat contributes little to the protective immune response, and mayactually complicate treatment in inducing allergenic and/or reactiveresponses.

In general the move in vaccine development has been towards biologicalagents that contain or promote only identified protein epitopes ofviruses that are deemed to induce positive, desirable T-cell and B-cellmediated immune responses. Typically the selected peptides are generally20-20 amino acid sequences. To determine the appropriate epitopecandidate multiple predictive algorithms have been developed employingtechniques such as nuclear magnetic resonance, X-ray crystallyography,mass spectrometry, phage libraries, recombinant cDNA libraries, andmimotopes. Computerized algorithms such as propensity scale,machine-learning algorithm, hybrid algorithm, ABCpred, ANN-BepriPred,HMM, BEDDPRo, SVM and PSSm may be used along with usage of databasescontaining known T cell-epitopes or peptides. A general discussion ofsuch techniques is set forth in Li, et al. Peptide Vaccine: Progress andChallenges, Vaccines 2014: 2:515-536.

In sum, the present vaccine approaches all make use of technologies thateither use inactivated or attenuated SARS-CoV-2 virus, or lead to themanufacture of proteins and amino acids that are unique to theSARS-CoV-2 virus.

The neutralization ability of an antibody on a virion is dependent onthe strength of interaction between the antibody and antigen and onconcentration. When the strength of antibody-antigen interaction isbelow a certain threshold, a phenomenon known as antibody-dependentenhancement (ADE) may be induced. It has been noted that when primateswere vaccinated with a modified vaccinia Ankara virus encodingfull-length SARS-CoV glycoprotein and challenged with SARS-COV, theprimates suffered from acute lung injury due to ADE. It has beenhypothesixed that ADE in coronavirus infection may be caused byconformation changes in the spike protein or a high mutation rate of thegene that encodes spike (S) protein. The least conservative amino acidsof SARS-CoV-2 appear to be the exposed fragments of the S-proteinincluding the receptor binding domain (RBD)(See, Ricke et al., MedicalCountermeasures Analysis of 2019-CoV and vaccine Risks forAntibody-Dependent Enhancement (ADE), SSRN Working paper Series, doi:10,21,2139/ssrn.3546070). There is some concern that SARS-CoV-2 vaccinesthat may initially demonstrate efficacy, may in the future lead to anADE catastrophe. Testing is underway to reduce such risk.

The present inventors propose herein a radically different approach fromthe 165 vaccine approaches discussed above, based on the discovery ofpre-COVID 19 T-cell cross-reactivity with the SARS-CoV-2 virus andreports of isolated large city (rather than by country or countynumbers) SARS-CoV-2 antibody levels and current daily infection anddeath rates in such cities. Taking note of Edward Jenner's work (Jenneroften referred to as the “Father of Immunology”) over a century agousing cowpox (indeed the term “vaccine” comes from the word “vacca”meaning cow) as a viral surrogate to prevent smallpox infections (ratherthan the then known immunization technique of variolation in whichmaterial from a small pox pustule was subcutaneously placed into anon-infected person), the present inventors propose a vaccine based oncommon coronaviruses associated with the common cold as a method ofproviding prolonged T-cell immunity to SARS-CoV-2 without the fear ofADE, particularly provided as a booster to directed SARS-CoV-2 epitopedirected immunizations. ADE is proposed to be reduced by expanding theT-cell immunity pool to more than just isolated spike proteins.

In a recent article by Grifoni et al., Targets of T Cell Responses toSARS-CoV-2 Cornonavirus in Humans with COVID-19 Disease and UnexposedIndividuals, Cell 181:1489-1501 (Jun. 25, 2020) using blood from 13samples garnered pre-COVID 19 from 2015 -2018 and comparing with 14convalescing COVID-19 patients, the authors identified regions of theSARS-CoV-2 virus that had cross-reactivity with other common circulatingcoronaviruses. Concern was raised that such cross-reactive immunitycould influence responsiveness to candidate vaccines.

Likewise, in a research paper by Matens et al., Selective andCross-Reactive SARS-CoV-2 T-cell Epitopes in Unexposed Humans, Science10.1126/Science.abd2871 (2020) the authors identified CD4+ T cells fromPBMC samples from unexposed subjects to SARS-CoV-2 that were collectedbetween March 2015 and March 2018 that were cross-reactive to SARS-CoV-2(82 out of 88). Blood from donors seropositive for common coldcoronaviruses (HCoVs) were utilized. Cross reactivity with HCoVs wasnoted with 142 epitopes derived from SARS-CoV-2 spike, N, nsp8, nsp12,and nsp13. When epitope homology was greater than 67% cross reactivitywas noted in 57% of cases (21 out of 37 samples). Strong responses wereseen directed to spike, ORF6, ORF3a, N, ORF8 and within Orfa/b, wherensp3, nsp12, nsp4, nsp6, nsp2 and nsp14 were prominently recognized. Theauthors noted that while it was plausible to hypothesize from the theirsmall sample comparisons that a pre-existing cross-reactivity HCoV CD4+T cell memory in some donors could be a contributing factor tovariations in COVID-19 patient disease outcomes, this is presentlyhighly speculative.

There are many reports of COVID-19 positive test results and totaldeaths reported daily by the CDC and WHO. The vast majority of thesereports are broken down by country and at best state/county within suchcountry. The present inventors have recognized that such totals do notdepict clearly what is happening in terms of the spread of SARS-CoV-2and potential cross immunities that may exist in one or more population.Antibody tests do not tell the whole story. T-cell immunity tests arenot widely available. Thus researchers at best have only been able tospeculate about possible cross-reactivities between coronaviruses, andthe extent that such cross-reactivity may have on a viable immuneresponse.

The inventors have rejected total numbers of cases and deaths in largeareas as providing any enlightment on previously acquired immunities.Instead, they have focused on city data, and reported numbers ofantibody seropositive percentages in such areas.

In particular, the inventors have found telling that although NYC hasbeen an epicenter of numerous peaceful and non-peaceful mass actionswith large numbers of people taking no heed of purported governmentalregulations as to distancing and masking, the number of deaths andreported cases has remained flat from July to August. One of the mostrecent studies of SARS-COV-2 IgG antibody in NYC, Reifer et al.,SARS-CoV-2 IgG Antibody Responses in New York City, DiagnosticMicrobiology and Infectious Disease, 98: 115128 (Jul. 1, 2020),indicates that nearly 44% of 28523 patients from the New York City areavisiting primary care providers and urgent care facilities in the NewYork City(the city and the surrounding boroughs of Kings (Brooklyn),Queens, New York (Manhattan), Bronx and Richmond (Staten Island) andsurrounding suburbs (Westchester, Rockland, Orange, Nassau and Sufflokcounties). In a CDC report updated Jul. 21, 2020, the CDC pegs theoverall prevalence of SARS-CoV-2 antibody seroprevalence of NYC betweenApr. 26, 2020-May 6, 2020 to be about 23.2 percent, with the lower boundbeing 19.9% and upper bound 26.3 percent. In Stockholm, as of May 29,2020 an on going study by the country's Public Health Agency reportsnearly 20 percent of Stockholm's population has antibody. See.Medicalxpress.com athttps://medicalxpress.com/news/2020-05-stockholm-virus-antibodies-sweden.html.In Stockholm, the reports of new COVID-19 deaths have plummeted sincethe high in April, and so have the number of reported cases. In London,as of May 29, 2020, 17 percent of Londoners were reported to testpositive for anti-SARS-CoV-2 antibodies by the Office for NationalStatistics (Burki, Talha Khan, Testing for COVID-19, The Lancet:Respiratory Medicine; 8(7) E63-64 (Jul. 1, 2020). Albeit that London hasalso been plagued with violent and non-violet protests with protestersnot socially distancing or masking appropriately in the summer of 2020,again London has seen a dramatic drop in the number of daily deaths andthe number of confirmed COVID-19 cases since July. In Dehli, wherenearly one in four residents (25%) of the Indian capital have shownantibodies to SARS-CoV-2, positivity ratios are now dramaticallyreducing as well as deaths. Nearly 50 percent of Mumbai slum clusterresidents are now reported to have antibodies to SARS-CoV-2, while only16 percent of the people in the more affluent parts do with highestpercentage of deaths occurring outside the slums. Mumbai slum dwellershave a much lower mortality rate of less than 0.05% than compared tocity proper dwellers in Mumbai. The increase in reported cases anddeaths in Mumbai is highly skewed toward city proper dwellers. Theinventors have rejected the hypothesis that the slightly youngerpopulation in the slums accounts for such disparity. Rather theyreasoned that people in the slums were much more prone to have beenexposed to other coronaviruses over their lifetimes, and that immunityfrom other coronaviruses, such as those causing the common cold, werethe basis for the dramatically lower mortality rate.

The present inventors hypothesize that the 20-25 percent antibodyfindings in respect of large modern disparate cities associated with adecrease in SARS-CoV-2 positivity and dramatically reduced deaths arenot happenstance or coincidental. Instead, they reasoned that the longlasting T-cell immunities associated with people affected by multiplecoronavirus common colds is adding to the protective effect of B-cellimmunity such that the population overall is heading to herd immunity.They note a number of studies that suggest CD4+ T-cells reactive toSARS-CoV-2 epitopes in approximately 50 percent of the population. Thesestudies alone do not provide a basis for determining whether suchcross-reactivities are indicative of robust immunity, or alternativelyimpeding responsiveness to candidate vaccines. The present inventorssuggest that the dramatic decreases in cases and deaths noted in largecities as they approach 25% of their population carrying antibodiesdemonstrate that the CD4+ T-cells reactivities noted by researchers areindeed indicative of robust immunities already native to the population.The fact that the Mumbai slums reached 50% antibody levels, may suggestthat such cell mediated immunity may not be perfected in all individualsto ward off SARS-COV-2, and that a population with 25% having antibodiesto SARS-CoV-2 may be sufficient to cause dramatic drops in death ratesand positivity when done in conjunction with other measures such associal distancing and masking.

They also note that studies on cross reactivity of CD4⁺ T-cells obtainedfrom persons before the SARS-CoV-2 epidemic, show cross reactivity withsamples that were obtained as long ago as 5 years ago. Their review ofthe data in this light led them to understand that common coldcoronaviruses could be used to provide long term cell-mediated immunityto SARS-CoV-2. They thus saw that the T-cell immunity effects noted withrespect to common cold viruses could be used as an adjuvant to anyprimarily B-cell immunity specifically directed SARS-CoV-2 vaccine, orone wherein T-cell immunity is not long lasting. Recent reports indicatethat antibodies to SARS-CoV-2 may disappear within as short as threemonths (See, Liu et al., Disappearance of antibodies to SARS-CoV-2 in aCOVID-19 patient after recovery, Clinical Microbiology and InfectionJul. 8, 2020 DPO; https//doi.org/10.1016/j.cmi.2020.07.009). Whileresident B-cell activity after SARS-CoV-2 infection still needs to bedetermined, such finding suggests that at least some vaccinations thatare directed to specific epitopes found on SARS-CoV-2 may not providelong term immunity in the line of years of protection. The presentinvention proffers an ability to provide long term T-cell mediatedimmunity in adjunct to any primarily short term B-cell immunity.

The present inventors also hypothesize that ADR is actually more relatedto a minimization of the profile of T-cells reactive (particular CD4+ Tcells) to a virus, rather than just reactivity due to human epitopesfound on the virus. Thus, their vaccine provides a booster to anyspecifically directed SARS-CoV-2 vaccine that is directed to a smallsubset of viral glycoproteins. The inventors hypothesize littlepotential reactivity due to common human-like epitopes found on theHCoVs particularly as humans have been exposed to such viruses for avery long period of time, maybe even millennia.

SUMMARY OF THE INVENTION

Accordingly, the invention herein provides in an embodiment a method ofusing inactivated human cold coronaviruses (HCoVs) vaccine, alone or asa booster, for the immunization against SARS-CoV-2 infections. PreferredHCoVs are selected from at least one of HCoV-NL63, HCoV-OC42, HCoV-299E,HCoV-OC43, HCoV-NL63 and HCoV-HKU1, and preferably selected from aplurality of such group, and most preferably 3, more preferably 4 ormore, and more preferably 5 or more. In a preferred embodiment thevaccine comprises HCoV virus envelope subunits. In a particularlypreferred embodiment the vaccine comprises HCoV virus envelope proteinsin a virus-like spheroid (VLS) (which may be approximately ovoid orspherical in shape). Such HCoV vaccine may be used as a booster, forexample, post-immunization with a vaccine designed to produce a specificSARS-CoV-2 protein, such as spike protein, to provide for longer lastingeffective T-cell memory. In an embodiment there is provided a method forinactivation of HCoVs, the method comprising the sequential steps ofexposure to copper atoms followed by hydrogen peroxide inactivation.Also provided is a method for separating genomic RNA and nucleoproteinsfrom the lipid protein/glycoprotein shell lipid to allow for formationof VLS by employment of a pH controlled lipophilic to hydrophilicsurface.

DESCRIPTION OF SPECIFIC EMBODIMENTS

In one embodiment there is provided a vaccine for improving T-cellimmunity, particularly CD4+ T cell immunity, to SARS-CoV-2, said vaccinebeing prepared by (a) providing a plurality of population of cells incell culture medium; (b) infecting each population of cells byinoculating the population of cells with the at least one of: HCoV-299E,HCoV-OC43, HCoV-NL63 and HCoV-HKU1 and incubating the inoculatedpopulation of cells to allow the virus in each cell culture medium toreplicate and propagate; (c) collecting the virus from each cell culturemedium; (d) purifying each of said virus from each cell culture; (e)inactivating each virus; and (f) preparing a pharmaceutical preparationfor inoculation having different antigens from at least two or more ofHCoV-299E, HCoV-OC43, HCoV-NL63 and HCoV-KHu1. In such embodiment, theantigens may comprise the whole virus, or part of such virus, from, forexample, the virus envelope or a protein associated with the virus. Thepopulation of cells may be Vero cells. Inactivation may be at least oneof beta propiolactone (BPL), hydrogen peroxide, formalin (formaldehyde)and copper. In one embodiment copper inactivation and hydrogen peroxideinactivation are sequential in order. The vaccine may further comprisean adjuvant one at least of which is selected from alum, Immodulon(IMM-101-containing a heat-killed whole cell Mobacterium obuense, arapidly dividing harmless spaprophyte), algammulin, monphosphoryl lipidA (MPL), resiquimod, muramyl peptide (MPD), N glycolyl dipeptide (GMDP),polylC, CpG oligonucleotide, aluminum salts, water in oil emulsion andoil in water emulsion.

Deactivation is preferably by exposing the HCoV at one time to copperions (particularly cupric) and at a distinct time hydrogen peroxide(sequential deactivation). Copper has a free electron in its outerorbital shell of electrons that allows it to easily take part inoxidation-reduction reactions. The copper pokes holes in the Coronaviruslipid coating which allows lower concentrations of other inactivatingagents such as beta-propiolactone, formalin or H₂O₂ to be used toinactivate the virus. The present inventors have recognized concomitantuse is sub-optimal. Whole killed virus, or protein/glycoprotein submitsof the viruses may be used in the making of the vaccine.

In another embodiment there is a combination vaccine for immunizationagainst SARS-CoV-2 infection comprising at least one unique epitope fromeach of a plurality in the group of HCoV-NL63, HCoV-OC42, HCoV-299E,HCoV-OC43, HCoV-NL63 and HCoV-HKU1, or the subgroup HCoV-299E,HCoV-OC43, HCoV-NL63 and HCoV-HKU1, of such unique epitope being uniqueto all other HCoVs in such group, or genetic instructions to make suchone unique epitope from each of viruses in the group, wherein the uniqueepitopes have epitope homology with SARS-CoV-2 of greater than or equalto 60%, more preferably greater than 70%, yet more preferably greaterthan 80%, and yet more preferably greater than 90%, or even morepreferably greater than 95%. In a preferred embodiment at least twounique epitopes from each virus of one, two, three, or four in the groupis combined, and in a more preferred embodiment at least three uniqueepitopes from each virus of one, two, three, or four in the group iscombined. The unique epitope in each case may be from the RNA virusenvelope. The unique epitopes may be limited to cross-reactivity withepitopes of SARS-CoV-2 in the SARS-COV-2 spike, N, nsp8, nsp12 andnsp13. The unique epitopes are preferably limited to epitopes foundassociated with the native HCoV-299E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1lipid bilayer. The unique epitopes may be naturally-derived,synthetically manufactured, or recombinantly produced. Such unique HCoVepitope vaccine may be used with any of the vaccines noted in the WHOWorld Health Organization, Draft Landscape of COVID-19 CandidateVaccines, 31 Jul. 2020, and similar technology based vaccines. In apreferred embodiment, such unique HCoV epitope vaccine is used as abooster such COVID-19 Candidate Vaccines set forth by the WHO 31 Jul.2020 to provide prolonged cell mediated immunity, particularly throughCD4⁺ T-cells.

Also provided in a vaccine for immunization against SARS-CoV-2 infectioncomprising at least one unique protein epitope, or genetic instructionsto make such one unique protein epitope, from a plurality of the groupof HCoV-299E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1, such unique proteinepitope being unique to all other HCoVs in such group, wherein theunique epitopes have epitope homology with SARS-CoV-2 of greater than orequal to 60%. The vaccine may comprises at least two unique proteinepitopes, or genetic instruction to make such two unique proteinepitopes, are selected from a plurality of the group of HCoV-299E,HCoV-OC43, HCoV-NL63 and HCoV-HKU1. The plurality of the group ofHCoV-299E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1 may be three HCoVs. Theunique protein epitopes have a homology with SARS-CoV-2 epitope of atleast greater than or equal to 50%, 60%, 67%, 70%, 80% 90%, or 95%. Thevaccine may be used as a booster to a SARS-CoV-2 specific vaccine.Preferably the unique protein epitopes have homology of at least 60%with at least one of SARS-CoV-2 spike, N, nsp8, nsp12 or nsp13, and yetmore preferably the SARS-CoV-spike protein.

Also provided is a vaccine for immunization against SARS-CoV-2 infectioncomprising at three or more unique protein epitopes associated with thelipid membrane from at least two of the group of HCoV-299E, HCoV-OC43,HCoV-NL63 and HCoV-HKU1, such protein epitopes being unique to all otherHCoVs in such group, wherein the unique epitopes have epitope homologywith SARS-CoV-2 of greater than or equal to 65%. The unique proteinepitopes may be associated with the lipid membrane. The vaccine of saidunique protein epitopes for each virus from the group may be in the formof lipid virus like spheroid particles. The lipid virus like spheroidparticles are formed by: (a) selecting copper foam with a copperskeleton of pores around 50 um; (b) immersing the copper foam an aqueoussolution of 0.03 M AgNO3 at room temperature; (c) treating the immersedsilver foam with a mixed ethanol solution containing HS(CH₂)₁₁CH₃ andHS(CH₂)₁₀COOH to form a treated copper foam; (d) drying the treatedcopper foam to form the final treated copper foam (FTCF); (e) runningenveloped virus from the group through untreated copper foam followed bythe treated copper foam in a pH 7-7.4 solution; (f) releasing materialscaptured by the final treated copper foam by directing solution with apH of 10.5-11 over said final treated foam; (g) separating out spheroidshaving a diameter between 90 nm-150 nm.

Coronaviruses are enveloped with a lipid bilayer, and are believed toinduce fusion of the viral envelope with the cell membrane to targetcells. Viral fusion glycoproteins are the key epitopes to induce themembrane fusion reaction that allows viral entry. U.S. Pat. No.6,455,050 to Aventis Pasteur Limited teaches techniques for obtainingviral envelope glycoproteins by means of an appropriate detergent (e.g.Triton X-100 or octylglucoside). Nucleopcapsids are taught to beremovable by centrifugation, with viral surface glycoproteins beingpurified from a glycoprotein enriched fraction by affinitychromatography, such as lentil-lectin and concanavlin A covalentlycoupled to cross-linked Sepharose or cellulosic microporous membranes.Viral surface glycoproteins are taught to be eluted from the column inthe presence of an appropriate competing sugar, such asmethyl-D-mannopyranoside, in the presence or absence of salt. Highlypurified glycoprotein preparations is said to be obtained in accord withsuch process (as judged by Coomassie blue or silver stained SDSpolyacrylamide gels).

Taught herein is another technique for isolating natural glycoproteinsof the lipid bilayer of coronaviruses from the nucleocapsid. Suchtechnique provides lipid bound glycoproteins that may be reannealed intovirus like spheroids (VLS). Such as system makes use of highlylipophilic surfaces that can by pH made to switch to highly hydrophilicsurfaces. Such technique may be used in conjunction with the with a HCoVvaccine or SARS-CoV-2 specific vaccine to provide more immunogenicity asseen with VLP (virus like particles). The present inventors propose thatADR is more likely when the antibody pool is more limited to just a fewproteins, such as those found on the spike protein of SARS-CoV-2, andthat irrespective of contrary thought, a more robust pool of antibodiesmay actually reduce ADRs. Such technique makes use of a switchablecopper foam having silver deposition followed by surface modificationwith a mixed solution of thiol containing carboxylic groups and methylgroups (HS(CH₂)₁₁CH₃ and HS(CH₂)₁₀COOH) to provide for pH reversibilitybetween a superhydrophobicity surface and a hydrophilicity surfaceprposed for removing oil from water. See, Liu et al., A Smart SwitchableBioinspired Copper Foam Responding to Different pH Droplets forReversible Oil-Water Separation, J. Mater. Chem. 2017, (5) 2603-2612, asexplained in Example 2 below. The VLS can be formed form at least one ofthe group of HCoV-299E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1, morepreferably at least two of the group, yet more preferably three of thegroup, or even more preferably form all of the viruses in the group. TheVLS spheroids may also be formed from SARS-CoV-2 itself. The VLSspheroids can be separated by chromatography, with cryo-electronmicroscopy being used to detect the same which generally should havediameters of 90-150 nm with glycoprotein protrusions/spikes. The VLSspheroids are then manufactured conventionally into vaccines, which, forexample, are used to provide immunity to SARS-CoV-2 infections.

EXAMPLE 1: HCoV VACCING FOR PROLONGING T-CELL IMMUNITY TO SARS-CoV-2

HCoV-299E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1 are isolated from lavagesamples, each from multiple persons suffering therefrom to cover thephylogenic tree. Different strains of each HCoV of each is plaquepurified and passaged once in Vero cells to generate a P1 stock. The P1stock is adaptively cultured, passed and expanded on Vero cells.Additional passages are performed to generate, for example, P2 to P5stocks. Growth kinetics are measured to assure efficient replication andto reach a peak titre of about 6 to 7 log₁₀ median tissue cultureinfections dose by 3 or 4 days post infection at a multiplicity ofinfection of, for example, 0.001 to 0.01 and temperatures between 33°and 37° C. Additional passages are performed to obtain the P_(final)stock (such as P10). Multiple P stocks are sequenced to assure geneticintegrity that might affect NAb epitopes. Whole genome of each strainand the P_(final) undergo deep sequencing analysis are undertaken toassure sequence homology of more than about 99.95%. P_(final) stock ispropagated in a culture of Vero cells (e.g. 50 liters) using the CellFactory system. Inactivation is brought about by inactivation with atleast one of beta propiolactone (BPL), hydrogen peroxide, formalin(formaldehyde) and copper. Purification is by depth filtration andmultiple (e.g. two) steps of chromatography to yield highly pure HCoVstock. Ultrafiltration, size exclusion chromatography and sucrosegradient centrifugation may be used in the purification process.B-propionolactone, for example, may be thoroughly mixed with harvestedviral solution at a ratio of 1:4,00 at 2° C.-8° C. Western blot analysisis used to show vaccine stock contains viral structural proteins. Two ormore, preferably three or more, of the purified and inactivatedHCoV-299E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1 are mixed with an adjuvantone at least of which is selected from alum, Immodulon(IMM-101-containing a heat-killed whole cell Mobacterium obuense, arapidly dividing harmless saprophyte), algammulin, monphosphoryl lipid A(MPL), resiquimod, muramyl peptide (MPD), N glycolyl dipeptide (GMDP),polylC, CpG oligonucleotide, aluminum salts, water in oil emulsion andoil in water emulsion, and pharmaceutical excipients to form anadministrable vaccine. Inactivation should be measured checking forimmunogenic response before and after immunization in animals. Dose maybe selected by looking at different doses and determining Nab levels at,for example, 7, 14 and 21 days in each of multiple dosing groups. Wholekilled virus, or protein/glycoprotein submits of the viruses may be usedin the making of the vaccine.

EXAMPLE 2: CORNAVIRUS ENVELOPE WITH ATTACHED GLYCOPROTEIN SEPARATIONFROM RNA AND NUCLEOPROTEINS

Copper foam with a copper skeleton of pores around 50 um is selected.The foam are immersed in an aqueous solution of 0.03 M AgNO3 at roomtemperature. The silver treated foam is then treated with a mixedethanol solution containing HS(CH₂)₁₁CH₃ and HS(CH₂)₁₀COOH to form theFinal Treated Copper Foam (FTCF). The enveloped virus is exposed tofirst to untreated copper foam followed by the treated copper foam in apH 7 solution. Untreated copper ions blast hole into the viral coating,while destroying RNA inside of the virus. Passage through the FTCF at pH7 makes the FTCF highly hydrophobic while at the same time highlylipophilic. The coronavirus flows through the pores of the copper foam.The coronavirus envelope is deposited along the FTCF at pH about 7-about7.4 with the RNA and nucleoproteins being washed away in the stream.Release of the attracted lipid from the envelope upon change of pH tomore basic pHs may form virus like spheres of natural glycoproteincovered lipid membrane (by changing the pH about the FTCF to about 10.5to no more than about pH 11 which makes the surface much less lipophilicand more hydrophilic). The pH is carefully controlled to avoidirreversible denaturation of the glycoproteins.

1. A method for providing a pharmaceutical immunogenic compositioncomprising the steps of: (a) providing a plurality of population ofcells in cell culture medium; (b) infecting each population of cells byinoculating the population of cells with the at least one of: HCoV-299E,HCoV-OC43, HCoV-NL63 and HCoV-HKU1 and incubating the inoculatedpopulation of cells to allow the virus in each cell culture medium toreplicate and propagate; (c) collecting the virus from each cell culturemedium; (d) purifying each of said virus from each cell culture; (e)inactivating each of said virus by at least one of beta-propiolactone(BPL), hydrogen peroxide, formalin (formaldehyde) and copper; and (f)preparing a pharmaceutical preparation for inoculation comprisingviruses consisting of two of HCoV-299E, HCoV-OC43, HCoV-NL63 andHCoV-HKU1 inactivated whole virus of steps (a)-(e).
 2. The method ofclaim 1 wherein the two of HCoV-299E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1consists of HCoV-229E and HCoV-OC43.
 3. The method of claim 1 whereinthe two of HCoV-299E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1 consists ofHCoV-229E and HCoV-NL63.
 4. The method of claim 1 wherein the two ofHCoV-299E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1 consists of HCoV-229E andHCoV-HKU1.
 5. The method of claim 1 wherein the two of HCoV-299E,HCoV-OC43, HCoV-NL63 and HCoV-HKU1 consists of HCoV-0C43 and HCoV-NL63.6. The method of claim 1 wherein the two of HCoV-299E, HCoV-OC43,HCoV-NL63 and HCoV-HKU1 consists of HCoV-OC43 and HCoV-HKU1.
 7. Themethod of claim 1 wherein the two of HCoV-299E, HCoV-OC43, HCoV-NL63 andHCoV-HKU1 consists of HCoV-NL63 and HCoV-HKU1.
 8. The method of claim 1wherein the cells infected at step (b) are Vero cells.
 9. The method ofclaim 1 further wherein the pharmaceutical composition prepared at step(f) comprises an adjuvant from one at least of alum, algammulin,monophosphoryl lipid A (MPL), resiquimod, muramyl depeptide (MPD), Nglycolyl dipeptide (GMDP), polylC, CpG oligonucleotide, aluminum salts,water in oil emulsion and oil in water emulsion.
 10. A method forproviding a pharmaceutical immunogenic composition comprising the stepsof: (a) providing a plurality of population of cells in cell culturemedium; (b) infecting each population of cells by inoculating thepopulation of cells with the at least one of: HCoV-299E, HCoV-OC43,HCoV-NL63 and HCoV-HKU1 and incubating the inoculated population ofcells to allow the virus in each cell culture medium to replicate andpropagate; (c) collecting the virus from each cell culture medium; (d)purifying each of said virus from each cell culture; (e) inactivatingeach of said virus by at least one of beta-propiolactone (BPL), hydrogenperoxide, formalin (formaldehyde) and copper; and (f) preparing apharmaceutical preparation for inoculation comprising viruses consistingof three of HCoV-299E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1 inactivatedwhole virus of steps (a)-(e).
 11. The method of claim 10 wherein thepharmaceutical composition prepared at step (f) further comprises anadjuvant from at least one of : alum, algammulin, monophosphoryl lipid A(MPL), resiquimod, muramyl depeptide (MPD), N glycolyl dipeptide (GMDP),polylC, CpG oligonucleotide, aluminum salts, water in oil emulsion andoil in water emulsion.
 12. The method of claim 10 wherein the cellsinfected at step (b) are Vero cells.
 13. The method of claim 10 whereinthe three of HCoV-299E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1 consists ofHCoV-229E, HCoV-NL63 and HCoV-HKU1.
 14. The method of claim 9 whereinthe three HCoV-299E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1 consists ofHCoV-OC43, HCoV-NL63 and HCoV-HKU1.
 15. The method of claim 9 whereinthe three of HCoV-299E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1 consists ofHCoV-229E, HCoV-OC43, and HCoV-HKU1.
 16. The method of claim 9 whereinthe three of HCoV-299E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1 consists ofHCoV-NL63, HCoV-229 E, and HCoV-OC43.
 17. A method for providing apharmaceutical immunogenic composition comprising the steps of: (a)providing a plurality of population of cells in cell culture medium; (b)infecting each population of cells by inoculating the population ofcells with the at least one of: HCoV-299E, HCoV-OC43, HCoV-NL63 andHCoV-HKU1 and incubating the inoculated population of cells to allow thevirus in each cell culture medium to replicate and propagate; (c)collecting the virus from each cell culture medium; (d) purifying eachof said virus from each cell culture; (e) inactivating each of saidvirus by at least one of beta-propiolactone (BPL), hydrogen peroxide,formalin (formaldehyde) and copper; and (f) preparing a pharmaceuticalpreparation for inoculation comprising viruses consisting of allof—HCoV-299E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1 inactivated whole virusof steps (a)-(e).
 18. The method of claim 17 wherein the pharmaceuticalcomposition prepared at step (f) further comprises an adjuvant from atleast one of : alum, algammulin, monophosphoryl lipid A (MPL),resiquimod, muramyl depeptide (MPD), N glycolyl dipeptide (GMDP),polylC, CpG oligonucleotide, aluminum salts, water in oil emulsion andoil in water emulsion.
 19. The method of claim 17 wherein the cellsinfected at step (b) are Vero cells.